Proteolytic silencing of cancer targets using engineered ubiquitin ligases

使用工程泛素连接酶对癌症靶标进行蛋白水解沉默

基本信息

批准号：
8584010
负责人：
MATTHEW P DELISA
金额：
$ 20.9万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-16 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8584010
关键词：
26S proteasome Ablation Acetylation Affinity Antibodies Antigens Antineoplastic Agents Apoptotic Area Binding Cancer Etiology Catalytic RNA Cell Death Cell Line Cell Proliferation Cells Chimera organism Consensus Disease Dissection Drug Targeting Ectopic Expression Engineering Enzymes Eukaryotic Cell Evaluation Event Family Gene Expression Gene Targeting Genes Genetic Engineering Goals Growth Human Inflammation Knock-out Life Lysine MAPK1 gene Malignant Neoplasms Mammalian Cell Measures Mediating Methods Methylation Mitogen-Activated Protein Kinases Modification Molecular Neoplasm Metastasis Oncogenes Oncogenic Pathway interactions Phenotype Phosphorylation Phosphotransferases Physiological Play Polyubiquitin Post-Translational Protein Processing Property Protein Conformation Protein Isoforms Proteins Proteomics RNA Recruitment Activity Reporting Research Role Route Signal Pathway Signal Transduction Site Somatic Cell Specificity Substrate Specificity System Technology Testing Therapeutic Therapeutic Intervention Time Tumor Cell Invasion Ubiquitin Ubiquitination United States National Institutes of Health base cellular targeting drug discovery glycosylation inhibitor/antagonist innovation intercellular communication interest member multicatalytic endopeptidase complex new technology novel novel therapeutics protein degradation public health relevance tool ubiquitin ligase ubiquitin-protein ligase

项目摘要

DESCRIPTION (provided by applicant): A variety of technologies such as gene targeting, ribozymes, antisense, or RNA-mediated interference (RNAi) have been developed for silencing of specific genes and assessing the physiological function of their products. Together, these approaches are enabling the discovery of novel drug targets in cancer and even the therapeutic knockdown of cancer-causing or cancer-promoting genes. However, for many applications it may be desirable to target specific protein conformations or post-translational modifications. In particular, protein phosphorylation plays a major role in numerous intracellular signaling pathways and represents a very active area of research in the quest for new therapeutic cancer targets. Unfortunately, modification-specific silencing is not possible using the above methods, all of which target gene expression. Therefore, this proposal seeks to develop a new functional proteomics technology based on antibody-like E3 ubiquitin ligase enzymes called "ubiquibodies" that are capable of silencing target proteins and their post-translationally modified isoforms. The innovativeness of this method lies in the creation of the ubiquibodies themselves, which are antibody-enzyme chimeras that combine the robust proteolytic activity of E3 ubiquitin ligases (E3s) with the affinity, specificity and modularity of intracellular single-chain Fv (scFv) antibodies. The resulting ubiquibodies are capable of recruiting the cell's robust protein degradation system to virtually any protein of interest. Since the scFv domain of a ubiquibody can be created to bind specific protein conformations or post-translational modifications such as glycosylation or phosphorylation, the technology has the potential for silencing of certain protein isoforms while sparing others. The long-term goal is to develop ubiquibodies as an innovative new protein silencing technology for functional analysis of cancer-associated proteins as well as for identification and pharmacological manipulation of cancer drug targets. Towards this goal, the following specific aims are proposed: first, under Aim 1, ubiquibodies will be engineered against the nonphosphorylated (inactive) and phosphorylated (active) isoforms of a well-studied target, ERK2, a member of the MAPK family whose members are well known to regulate several physiological and pathological phenomena including inflammation, apoptotic cell death, oncogenic transformation, tumor cell invasion, and metastasis. Second, under Aim 2, the consequences of ectopic expression of anti-ERK2 ubiquibodies will be examined with respect to ERK2 signaling as well as growth and proliferation of mammalian cells. It is anticipated that isoform-specific silencing will help to uncover valuable information about ERK's role in signaling and cell proliferation. More broadly, this new protein knockout system will provide a simple and efficient tool for creating artificial E3 ligases that enable: (i) dissection of diverse functional properties of cellular proteins in somatic cells; (ii) evaluation of whether specific cellular protins are valid targets for therapeutic intervention; and (iii) ablation of intracellular protein targetsas an effective therapeutic strategy for certain human malignancies such as cancer.

描述（由申请人提供）：已经开发了多种技术用于沉默特定基因并评估其产物的生理功能，例如基因靶向、核酶、反义或RNA介导的干扰（RNAi）。这些方法共同促进了癌症新药物靶点的发现，甚至可以治疗性敲低致癌或促癌基因。然而，对于许多应用来说，可能需要针对特定的蛋白质构象或翻译后修饰。特别是，蛋白质磷酸化在许多细胞内信号传导途径中发挥着重要作用，并且代表了寻求新的癌症治疗靶点的非常活跃的研究领域。不幸的是，使用上述方法不可能实现修饰特异性沉默，所有这些方法都靶向基因表达。因此，该提案旨在开发一种基于抗体样 E3 泛素连接酶（称为“ubiquibodies”）的新功能蛋白质组学技术，该技术能够沉默靶蛋白及其翻译后修饰的异构体。这该方法的创新之处在于泛素体本身的创建，它们是抗体-酶嵌合体，将 E3 泛素连接酶 (E3s) 强大的蛋白水解活性与细胞内单链 Fv (scFv) 抗体的亲和力、特异性和模块化相结合。由此产生的泛在体能够将细胞强大的蛋白质降解系统招募到几乎任何感兴趣的蛋白质。由于可以创建泛在体的 scFv 结构域来结合特定的蛋白质构象或翻译后修饰（例如糖基化或磷酸化），因此该技术具有沉默某些蛋白质的潜力同工型，同时不伤害其他同工型。长期目标是开发泛在体作为一种创新的蛋白质沉默技术，用于癌症相关蛋白质的功能分析以及癌症药物靶标的识别和药理学操作。为了实现这一目标，提出了以下具体目标：首先，在目标 1 下，将针对经过充分研究的靶标 ERK2 的非磷酸化（无活性）和磷酸化（活性）亚型进行工程改造，ERK2 是 MAPK 家族的成员，其成员众所周知，其调节多种生理和病理现象，包括炎症、细胞凋亡、致癌转化、肿瘤细胞侵袭和转移。其次，在目标 2 下，将检查抗 ERK2 泛素体异位表达对 ERK2 信号传导以及哺乳动物细胞生长和增殖的影响。预计异构体特异性沉默将有助于揭示有关 ERK 在信号传导和细胞增殖中的作用的有价值的信息。更广泛地说，这种新的蛋白质敲除系统将为创建人工 E3 连接酶提供简单而有效的工具，从而能够：（i）解剖不同的功能体细胞中细胞蛋白的特性； (ii) 评估特定细胞蛋白是否是治疗干预的有效靶标； (iii) 消除细胞内蛋白质靶标作为某些人类恶性肿瘤（如癌症）的有效治疗策略。